Cerebral Cavernous Malformations (CCM) are vascular lesions of the central nervous system consisting of closely-packed, grossly-enlarged capillary-like vessels. CCM lesions develop through time from single dilated vessels into complex, multicavernous lesions and have a propensity for bleeding leading to hemorrhagic stroke. CCM may occur sporadically or follow an autosomal dominant pattern of inheritance. For the inherited condition, three loci have been mapped and the causative genes (CCM1, CCM2, and CCM3) have been identified. While the functions of these novel gene products have begun to be elucidated, there is no clear connection to their influence on lesion genesis or the mechanism for lesion progression. I hypothesize that CCM lesions arise due to a second-site somatic mutation leading to molecular homozygosity at the CCM gene locus. The two-hit hypothesis for CCM lesion genesis is consistent with the repeated observational difference between sporadic and familial CCM; multiple lesions develop in patients with familial CCM cases, but single lesions develop almost exclusively in sporadic cases. A similar pattern of incidence in familial and sporadic cases of retinoblastoma led to the development of a two-hit model by Knudson, in which both allelic copies of a tumor suppressor gene must be mutated to yield tumor formation. The two-hit hypothesis makes specific predictions for the mutational events leading to tumorogenesis in cancer. I propose to test these predictions for a non-cancer model; that of CCM using our collection of archived tissue samples. The two-hit hypothesis predicts that each class of CCM lesions, those with inherited mutations in CCM1, CCM2, or CCM3 will show somatic mutation within the lesion that is biallelic to the germline mutation. Additionally, the two-hit hypothesis predicts that sporadic CCM lesions will routinely harbor two distinct biallelic somatic mutations within one of the CCM genes. A key prediction of the two-hit hypothesis regarding the molecular genetic origins of multiple lesions in familial cases is that multiple lesions arise due to independent somatic mutational events. The following aims have been designed to comprehensively study the two-hit hypothesis using clinically-relevant mature human CCM lesion samples. [unreadable] 1a. To investigate the two-hit hypothesis in FAMILIAL cases of CCM. [unreadable] 1b. To validate the two-hit hypothesis in cases of familial CCM with MULTIPLE lesions [unreadable] 2. To investigate the two-hit hypothesis in SPORADIC cases of CCM. [unreadable] Through successful completion of these aims I intend to more fully understand the underlying fundamental biology of CCM, an inherited form of stroke, to impact future biologically-based therapy. [unreadable] [unreadable]